The objective of the project is to gain insight into the molecular mechanism of dinitrogen (N2) reduction catalyzed by the metalloenzyme nitrogenase. Our goal in this proposal is to develop a unique experimental methodology that will enable a direct examination of substrate reduction. Measuring competitive 15N kinetic isotope effects (KlEs) will allow examination of the change in nitrogen bond order along the reaction coordinate. These experiments may enable the first direct investigation of the dinitrogen reduction step within the nitrogenase complex kinetic cascade. D20 and D2 effects (D KIEs) on the 15N KIEs will enable examination of the reductive protonation of N2 and estimation of intrinsic KIEs. Triple labeling experiments (15N15N /15N14N/14N14N) will further assist the elucidation of the intrinsic mechanism.The initial experiments will be conducted with the enzyme from Azotobacter vinelandii whose crystal structure is available. The experimental findings will be used to reevaluate various theoretical approaches that were based on the metallic cluster coordinates from that crystal structure, and will support or invalidate proposed mechanistic models.The long-term plans include an investigation of various forms (mutants) of the enzyme with altered reaction site environments and nitrogenases from various organisms and non-enzymatic catalysts. This study may lead to a new and unique understanding of the mechanism by which nitrogenase catalyzes dinitrogen reduction and can have a critical impact in the field of nitrogen fixation and metalloenzymes in general.